Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDA) has the worst five-year survival rate of any major cancer. The lethality of PDA is largely due to a lack of effective treatment options. Several barriers in PDA treatment are conferred by the presence of tumor associated macrophages (TAMs), which are highly represented in these tumors. TAMs have been shown to drive resistance to chemotherapies, and are strongly immunosuppressive toward cytotoxic T-cells. This immune suppression renders PDA refractory to immunotherapy treatments which have proven effective in other solid tumors. Consequently, the elimination or reprogramming of TAMs offers multiple potential avenues to provide a much-needed breakthrough for the treatment of patients with PDA. An epithelial-immune crosstalk signaling mechanism leading to immunosupression of cytotoxic T-cells by PDA cells has been identified. TAMs are polarized through cytokines released from PDA cells, and TAMs in turn release ligands which activate mitogen activated protein kinase (MAPK) pathway in cancer cells, leading to expression of checkpoint ligands. MAPK activity drives the altered metabolic state of PDA cells, including upregulation of lactate production. Lactate in the tumors can act to polarize TAMs, thus the metabolic byproducts of PDA cells can determine the polarization state of TAMs. Preliminary data suggests that TAM polarization is, at least in part, driven by PDA metabolic byproducts. These data also show this polarization of TAMs results in an immune-suppressive arginine metabolic program and release of several metabolites confer chemoresistance to the PDA cells. Metabolic polarization allows for more TAMs in the tumor, which release more signaling molecules to activate MAPK signaling in PDA cells, further propagating this feed-forward loop. The working hypothesis of this proposal is that interruption of the signaling and metabolic mechanisms driving this crosstalk pathway will improve pancreatic cancer therapy. This will be examined in two parts. First, metabolic weaknesses of TAMs programmed by PDA cells will be identified, and the functional consequences of targeting these features will be measured against TAM-PDA crosstalk mechanisms (Aim 1). Metabolic weaknesses will be identified through transcriptomics, metabolomics, and flux measurement techniques. The effect of metabolic inhibitors on PDA-TAM crosstalk will then be examined in co-culture systems. Secondly, the metabolism of TAMs will be targeted in combination with drug treatment to examine the impact of these processes on PDA tumors (Aim 2). These combination treatments will first be examined measuring PDA viability using in vitro co-culture systems. The results of the in vitro assays will then be verified using syngeneic xenograft tumor models coupled with genetic and pharmacological modes of macrophage depletion and/or reprogramming.